Analysis of non-coding RNA in granulocyte-macrophage colony-stimulating factor (GM-CSF) transgenic mice. (A) Northern blot analysis of GM-CSF mRNA expression probed with 1.3 Sal I-Eco RI gene fragment, and 3' non-coding RNA expression probed with a 1.0 kb Bgl II-Hind III fragment. The lower panel shows methylene blue staining of RNA. (B and C) Strand-specific reverse transcription and PCR amplification of regions A, B, C, and J using the primers listed in Table 2, and T cell RNA prepared before and after stimulation. Line C42 contains 6 copies of a 130 kb segment of the GM-CSF locus whereas line A127 contains 4 copies of a 10.5 kb Xho I-Hind III fragment of the GM-CSF locus. PCR reactions employed reverse transcriptase and PCR primers (PRT), primers but no reverse transcriptase (-RT), or reverse transcriptase without PCR primers. Bars depict PCR amplicons. (D) Analysis of siRNAs in transgenic T cells. Either total RNA (lanes 1 to 4), or fractionated small RNA (lane 5) was subjected to polyacrylamide gel electrophoresis, transferred to nylon membranes and hybridised with the probes described in the methods section. RNA was prepared from cells either 1 (lanes 2 and 3), 3 (lane 3) or 7 (lanes 4 and 5) days after initial stimulation of spleen cells with ConA, which was removed after the first 2 days. The upper panel encompasses the region of the filter where siRNAs should migrate (~20-25 bp).